Studying the metabolome

Two generalised experimental strategies are applied in metabolomic studies depending on the biological question being investigated. These strategies are defined as hypothesis-generating (or discovery) and hypothesis-testing (or validation) studies and differ in the number of metabolites investigated, the experimental design, the analytical platforms and data analysis methodologies applied [1]. Hypothesis-generating studies apply an untargeted approach, typically called metabolic profiling, to investigate large numbers of metabolites. In hypothesis-testing studies, a targeted analytical method is applied that focuses on a limited number of metabolites.

Untargeted analysis/Metabolic profiling

In hypothesis-generating (or discovery) studies, experiments are designed and performed to generate an overview of the metabolome; samples are compared in order for differences to be identified. These studies apply a data-driven approach to enable the construction of hypotheses at the end of the study. Although an ideal study would allow detection of all metabolites, the diversity of concentrations and physicochemical properties of metabolites means that this is not achievable. Instead metabolic profiling is performed where hundreds of metabolites are detected in a single study with relative quantification data reported. Statistical analysis of datasets provides information on metabolic differences related to genetic, biological or environmental perturbations.

Targeted analysis

This alternative strategy is based on the measurement of a specific metabolite or metabolites defined at the start of the study. This may include validation of metabolite changes observed in an ‘untargeted’ metabolomics study. Typically, a small number of metabolites of interest are investigated, with the ability to report absolute quantification data (concentration of analyte per unit of sample), applying analytical platforms that generate results with higher sensitivity and specificity compared to hypothesis-generating studies.